Phylogenetic Analysis of Hemagglutinin Genes of H9N2 Avian Influenza Viruses Isolated from Chickens in Shandong, China, between 1998 and 2013

Since H9N2 avian influenza virus (AIV) was first isolated in Guangdong province of China, the virus has been circulating in chicken flocks in mainland China. However, a systematic phylogenetic analysis of H9N2 AIV from chickens in Shandong of China has not been conducted. Based on hemagglutinin (HA) gene sequences of H9N2 AIVs isolated from chickens in Shandong of China between 1998 and 2013, genetic evolution of 35 HA gene sequences was systematically analyzed in this study. Our findings showed that the majority of H9N2 AIVs (21 out of 35) belonged to the lineage h9.4.2.5. Most of isolates (33 out of 35) had a PSRSSR↓GLF motif in HA cleavage site. Importantly, 29 out of these 35 isolates had an amino acid exchange (Q226L) in the receptor-binding site. The substitution showed that H9N2 AIVs had the potential affinity to bind to human-like receptor. The currently prevalent H9N2 AIVs in Shandong belonged to the lineage h9.4.2.5 which are different from the vaccine strain SS/94 clade h9.4.2.3. Therefore, the long-term surveillance of H9N2 AIVs is of significance to combat the possible H9N2 AIV outbreaks.


Introduction
Since H9N2 subtype avian influenza virus (AIV) was first isolated in Guangdong province of China in 1994, this subtype influenza virus has quickly spread to other areas of China and become prevalent in poultry [1][2][3]. H9N2 AIV infections in chicken flocks have led to massive economic losses due to egg production decline or high mortality related with coinfection with other pathogens [4][5][6].
Although there is no evidence for human-to-human transmission of H9N2 AIV, several human infections with H9N2 AIV have been reported [7][8][9][10][11]. It is noteworthy that the number of humans infected by H9N2 AIV in serological surveillance, especially poultry workers, was much higher than that of the confirmed cases [12,13]. H9N2 AIV is able to reassort with other subtypes of influenza virus, including H6N1, H6N2, H5N1, and H7N9. The increased risk of animalto-human spread with avian viruses that have the H9N2 internal genes has posed a greater pandemic threat [14][15][16].
Since the late 1990s, vaccination has been carried out in China to prevent and control H9N2 AIV infection in chicken flocks. Commercial vaccines did not provide complete protection for the endemic H9N2 AIVs in China. Up to the present time, H9N2 virus has regularly been found in vaccinated chickens in Shandong of China [1,17,18]. Therefore, this study was designed to perform a comprehensive phylogenetic analysis of H9N2 AIVs from chickens in Shandong of China to provide an important guidance for the effective control of H9N2 AIV infection.

Phylogenetic Analysis.
Multiple sequence alignment was conducted using ClustalW [19]. The phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates using MEGA 5.05 [20].

Antigenic
Analysis. Antigenic analysis of H9N2 AIV was conducted using hemagglutination inhibition (HI) to assess antigenic relationship between the emerging H9N2 AIVs and the vaccine strain SS/94 [21]. According to the previously published work [22], 6-week-old specific pathogen-free (SPF) chickens were used to produce polyclonal antibodies against 7 H9N2 AIVs, including 6 isolates between 2012 and 2013, and the vaccine strain SS/94.   (Figure 1).

Important Site Analysis of Deduced AA Sequences.
The cleavage sites of precursor HA protein of H9N2 AIVs showed two different motifs in this study. 33 H9N2 AIVs isolated between 1999 and 2013 had a PSRSSR↓GLF motif, which can meet the characteristic of low-pathogenic avian influenza virus (Table 3). AA residues at receptor-binding site of HA protein were conserved among the 35 H9N2 AIVs. Only one substitution was found in the position A190V(T) (H3 numbering is used throughout the paper) ( Table 3). On the right edge of receptor-binding pocket, AA sequences were conserved and no substitution was found in this study. AA residues on the left edge of receptor-binding pocket had a substitution Q227L (M), and most isolates ( (Table 3).

Discussion
Vaccination is an effective way to prevent and control the spread of H9N2 AIVs, but the current vaccine used in Shandong is still prepared from the isolates in the early 1990s, and their protective efficacy has been decreasing [3,18]. HI test in this study showed that commercial vaccine strain SS/94 has not provided robust protection against the emerging H9N2 AIVs isolated in Shandong, China.
Previous investigation about phylogenetic analysis of HA genes of H9N2 AIVs isolated in China between 2008 and 2011 demonstrated that the lineage h9.4.2.5, as an emerging lineage, has become one of the dominant clades [22,25]. Similarly, our findings showed that the lineage h9. 4.2.5 was predominant between 2009 and 2013 in Shandong. The vaccine strain SS/94 belonged to the distinct lineage h9.4.2.3. Antigenic analysis also showed that the emerging viruses between 2012 and 2013 were antigenically different from the vaccine strain SS/94. Given the above results, it is of utmost importance to select novel candidate vaccine strains to combat the emerging H9N2 AIVs in Shandong of China.
On the left edge of receptor-binding pocket, most isolates (4 out of 6) between 2012 and 2013 had methionine (M) at position 227. But the vaccine strain SS/94 had glutamine (Q) at the same position. 33 H9N2 AIVs isolated between 1999 and 2013 had a PSRSSR↓GLF motif in HA cleavage site; the vaccine strain SS/94 had a PAGSSR↓GLF motif. Additionally, 29 out of 35 H9N2 AIVs had leucine (L) at position 226, including all of the isolates between 2005 and 2013. The substitution indicated that the emerging H9N2 AIVs can preferentially bind to NeuAca2,6-Gal linkage and may have higher virulence [3,26].

Conclusion
Taken together, genetic disparity between the emerging H9N2 AIVs and the current vaccine isolates should be taken into consideration when a control policy is established. In addition, the long-term surveillance of H9N2 AIVs is of significance to combat the possible H9N2 AIV outbreaks in chicken flocks.

Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.